Mesenchymal stem or stromal cells (MSCs) are multipotent cells of embryonic mesodermal origin, with a fibroblast-like morphology. These cells can differentiate into adipocytes, osteocytes, chondrocytes, neural lineage cells, and myocytes among other cell types depending on the stimuli and culture conditions. Although the plasticity of hMSCs and their role in tissue repair and regeneration have been extensively studied, it is their immunological trophic property that has gained the most interest recently [50-51]. Human mesenchymal stem cells have been isolated from a variety of tissues. The most frequently used source of MSCs is the bone marrow (BM). However, the isolation procedure is extremely invasive. To avoid the invasive isolation procedures, tissues such as human umbilical cord and placenta have been considered as good candidates since they are normally discarded after labor. The isolation of hMSCs from umbilical cord or placenta has proven to be efficient by previous studies [49].
MSCs are a subpopulation of a more complex cell composition of stromal cells contained in mesenchymal tissue. Due to the heterogeneous nature and the absence of known biomarkers specific for mesenchymal stem cells, it is a challenging task to define MSC phenotypes and characteristics [52-54]. The molecular components responsible for MSCs functionalities, in particular, those on the plasma membrane, remain largely unknown. In addition, lack of specific cell surface markers renders the cell culture at potential contamination risk with other cell types, in particular, those mature stromal cells such as fibroblasts, which, conversely, are abundant in mesenchymal tissue [52-54]. In the process of isolation of MSCs from placenta-derived tissues, non-MSCs, including fibroblasts, placenta-derived epithelial cells, and placenta-derived reticular cells, often co-exist with MSCs during the in vitro cultivation. In particular, fibroblast is the main source of contamination.
Fibroblasts are considered mature mesenchymal cells that are particularly abundant in the connective tissue. Therefore, these cells are the most frequent contaminating cell phenotype present in many cell culture systems. Not only is it difficult to apply techniques which successfully eliminate fibroblasts from a culture, it is also particularly complex to distinguish MSCs from fibroblasts in the same culture. Fibroblasts and MSCs have an extremely similar morphological appearance; they both proliferate well and share many identical cell surface markers [55, 56]. MSC are currently defined as plastic adherent, multipotent fibroblast-like cells expressing CD73, CD90, CD105 and negative for the hematopoietic markers CD14, CD34 and CD45 by the International Society of Cellular Therapy (ISCT). However, these properties and markers are also shared by fibroblasts. The current definition suggested by ISCT is thus incapable of distinguishing MSC from generic fibroblasts. Until now, the best way to distinguish MSCs from fibroblasts is based on the analysis of the functional properties of these two kinds of cells; MSCs retain multipotent stemness and immunomodulation capacity, while fibroblasts seem more limited in both of these functional properties.
Since Friedenstein's pioneering work in identification of MSCs [48], there are no distinct differences in culture-derivation methodology, morphology, and gene expression signature that consistently and unequivocally distinguish ex vivo culture-expanded MSC from fibroblasts [57-60]. Presently, there is no accepted criterion or single cell-surface marker for separating the MSCs from fibroblasts. Due to the fact that fibroblast is the common contaminant cell population in MSC culture when derived from placenta, a novel surface protein as a biomarker to distinguish MSCs from fibroblasts is crucial to ensure the homogeneity of primary culture of placenta-derived MSCs.
The human erythropoietin-producing hepatocellular (Eph) receptors include transmembrane proteins comprising the largest family of receptor tyrosine kinases (RTKs). The first identified functions of Eph receptors were their roles in the complicated and sophisticated mechanism in axon guidance [4]. Eph receptors are now known to regulate a wide range of cell-to-cell communication events involved in cell positioning and tissue patterning during embryonic development and pathological conditions such as cancer and vascular complications [1-5]. In addition, these receptors are important regulators of specialized cell functions in synaptic plasticity, insulin secretion, bone remodeling, epithelial homeostasis, as well as inflammatory and immune responses [1, 2, 6]. They are expressed by a wide variety of cell types such as neurons, vascular cells, epithelial cells, inflammatory cells, immune cells, and tumor cells including cancer stem cells [7-10].
EphA2 gene belongs to the Eph receptor subfamily of the protein-tyrosine kinase family. Previous studies have been implicated EphA2's functions in mediating developmental events, particularly in the nervous system [4]. During development, EphA2 functions in distinctive aspects of pattern formation and subsequently in development of several fetal tissues, including vasculogenesis, neural tube development, axial mesoderm formation, early hindbrain development, neuron differentiation, regulation of cell migration, bone remodeling through regulation of osteoclastogenesis and osteoblastogenesis, mammary gland epithelial cell proliferation and branching morphogenesis during mammary gland development [11]. In particular, EphA2's role in nervous system embryonic development is well-defined [12], including the process by which neurons send out axons to reach the correct targets.
Roles of Eph receptors have been implicated in stem cell biology only recently, both during embryonic development and in the adult stem cell niche. Eph receptors are expressed in most adult stem cell niches. Stem cells are located in specialized microenvironments, niches, defined as the combination of cellular and microenvironmental determinants orchestrating the self-renewal and differentiation of stem cell pools within specialized tissue locations. The expression of Eph receptors and ephrin ligands during embryogenesis and tissue homeostasis is consistent with their involvement in stem cell regulation during development and in adult tissue homeostasis [13, 15]. It has been suggested Eph/ephrin system carry out a spatio-temporal regulatory function in the balance between stem cell quiescence, self-renewal and differentiation [14]. However, the mechanism of Eph in stem cell niche maintenance and its role in regulating stem cells are not well understood. EphA2 is highly expressed in embryonic stem cells [16]. Nevertheless, the majority of the EphA2 functional studies in stem cells have been focused on the nervous system. EphA2 is highly expressed in CNS, including precursors in neuronal and glial lineages [12, 15]. Recent studies provide evidence that ephrin-A1 promotes the motility of EphA2-positive cardiac stem cells, resulting in enhanced regeneration and cardiac function after myocardial infarction [17]. Beside these findings, the expression profile and functions of EphA2 in stem cell science are not yet well determined.
Eph receptors and ephrin ligands regulate both self-renewal of stem/progenitor cells and tumor progression [14]. High-degree similarity between untransformed stem/progenitor cells and cancer cells is also acknowledged. In recent years the concept of numerous cancers harboring a “cancer stem cell” compartment, comprising up to 25% of the cancer cells population, has been described [14]. These cells have been defined as tumor-propagating cells (TPCs) for their ability to induce tumors in animal hosts, self-renew and give rise to more differentiated cells in expanding tumor cell mass [14]. Recently, Eph/ephrin signals were linked to the regulation of cancer cell dedifferentiation and stem-like properties [9, 18, 19]. However, it is to be noted that cancers stem cells are actually not (multipotent) “stem cells” as generally referred to in the relevant art.
The overexpression of Ephs coupled with the down-regulation of the specific ephrin ligands has been reported in several cancers and associated with tumor aggressiveness and higher grades [19-22]. EphA2 expression is elevated in breast, ovarian, and lung cancer, as well as in glioma and melanoma, and high levels of EphA2 are correlated with poor patient survival [20, 23-29]. However, the roles and the expression of Eph receptor in cancer cells are absolutely context-dependent. A reverse expression pattern has also been observed in some tumors including breast, colorectal cancer, and acute lymphoblastic leukemia, where low Eph receptor expression through epigenetic silencing or mutations correlate with poor prognosis [30]. In the study of transcription profiling by array of human adrenocortical carcinomas, adenomas and healthy adrenal cortex tissues, EphA2 expression was down-regulated in human adrenocortical tumor tissues when compared with healthy adrenal cortex tissues [31]. Hence, although the expression patterns of certain Ephs and ephrins can serve as prognostic markers in many tumorigenesis cases, a reverse phenomenon in substantial amount of study reports was also observed. Expression of Eph/ephrins is critically cell/tumor-context-specific and context-dependent.
Recent studies on glioblastoma (GBM) showed that tumors harboring a large subpopulation of TPCs demonstrate increased expression of EphA2 and EphA3. The EphA2 receptor is overexpressed in human glioblastoma cancer stem cells (CSCs), and EphA2 expression positively correlated with the size and tumor-initiating ability of the CSCs in this specific type of tumor [9]. These Eph receptors regulate central nervous system development whereas their deregulated expression and somatic mutations are associated with growth, progression and metastasis of nervous system tumors [32-36].
On the other hand, ligand-dependent activation of EphA signaling possessing a tumor-suppressive effect in GBM, colorectal, breast, prostate and skin cancer were also reported [27, 38-43]. In substantial GBM studies, activation of EphA2 kinase by ephrinA1 were reported to have an anti-proliferative effect, possibly through down-regulation of EphA2 and FAK activities [27, 38, 44]. EphA2 knockout mice display increased tumor cell proliferation and ERK phosphorylation [45]. Ligand stimulation of EphA2 also attenuates EGF-mediated ERK phosphorylation, which correlates with reduced cell proliferation and migration [46, 47]. Altogether, interestingly, these findings support the tumor growth-suppressive and invasion-suppressive EphA2/ephrinA1 signaling. The result of the ephrin-Eph interaction is remarkably divergent in different contexts.
The research paper published by Vescovi's group in 2012 [9] demonstrating that (1) stem-like tumor-propagating cells (TPCs) in hGBMs highly express EphA2 receptors, (2) high EphA2 expression supports the undifferentiated state and self-renewal in TPCs, (3) TPC content and tumorigenicity are higher in EphA2[High] than EphA2[Low] hGBM cells. Despite the observed facts set forth above, EphA2[Low] hGBM still possess significant tumor-initiating capability. One could argue if EphA2 represents a true TPC marker even in hGBM, let alone in a different tumor or a different type of cells. In other words, one skilled in the art would not have acknowledged that EphA2 is a specific and universal marker for TPCs, much less a specific and universal marker for multipotent stem cells. The same group also filed a patent application claiming the use of EphA2 as a cell surface marker for the identification and the isolation of a stem cell, preferably a mammalian stem cell, more preferably a human or mouse stem cell [37, EP 2733206 A1]. However, in view of that Vescovi's study entirely and only focused on human glioblastomas (hGBMs) and the facts that EphA2[Low] hGBM still possess significant tumor-initiating capability, one skilled in the art would have in no way recognized that EphA2 is a specific marker competent in identifying multipotent stem cells. Further, Vescovi is also silent as to how to distinguishing multipotent stem cells in a primary culture of cells derived from a placenta-related tissue.